Detection and analysis of influenza virus

ABSTRACT

An assay comprising more than one primer pair and more than one detection probe, a low copy number synthetic amplicon corresponding to each of the primer pairs. The assay can detect and distinguish between various sub-types and strains of an influenza virus using any suitable nucleic acid amplification technique. Related kits and methods are also described.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119 of U.S.provisional application No. 60/819,000, filed Jul. 7, 2006, which ishereby incorporated by reference in its entirety.

BACKGROUND

Rapid detection and typing of influenza virus and identification of itsvarious strains is critical to identification and control of a potentialhuman pandemic. Influenza virus is composed of eight single-stranded RNAmolecules (HA, NA, PB2, PB1, PA, NS, M, NP) that code for elevenspecific proteins. The RNA for the matrix protein (M) is relativelyconserved and is therefore used to detect and distinguish a Type Avirus. M can also be used to detect and distinguish H5N1.

The hemagglutinin protein (HA) and neuraminidase protein (NA) aregrouped into 16 and 9 subtypes, respectively, both have high sequencevariability even within subtypes and thus provide an effective means ofmonitoring changes that might occur in a virus. The HA protein protrudesfrom the surface of the virus and allows it to attach to a cell to beginthe infection cascade. The NA protein is also located on the surface ofthe virus and allows the release of new particles within the infectedcell.

Currently the Eurasian H5N1 virus infects only the lower lungs in humanand is therefore less readily transmitted human-to-human than annualstrains of human influenza that infect the upper respiratory track. But,mutations within the HA and NA RNAs are frequent and alter viralinfectivity and lethality in different hosts and their tissues. Inaddition, gene assortment among the different viral subtypes is anothervery worrisome feature of influenza and could result in recombining RNAsequences for high infectivity in humans with high lethality.

SUMMARY

Accordingly, there is a need for an informative influenza assay that canbe performed in the field, i.e., at the point of care (“POC”). Moreover,in order to save both time and money it will also be important to makePOC assays compatible with more extensive laboratory analysis, such assequencing of, for example, HA and NA. In this way, the evolution of aviral disease and viral genomics can be analyzed in real-time.

One embodiment is directed to an assay comprising a plurality of primerpairs, a plurality of probes, and a low copy number synthetic ampliconcorresponding to each of the plurality of primer pairs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an embodiment of LATE PCR (left) and providesfluorescence curves produced by LATE PCR.

FIG. 2 is an agarose gel showing four sets of reactions, performed intriplicate, each reaction using the same Excess Primer (thick arrow),plus a different own Limiting Primer (thin arrow). The meltingtemperature of the four different Limiting Primers increases from leftto right and the annealing temperature used for each set of reactions is2° C. below the melting temperature of the Limiting Primer.

FIG. 3 shows agarose gels of identical LATE-PCR without ELIXIR (left)and with ELIXIR (right). The replicate reactions are prepared using fourdifferent preparations of commercially available Taq polymerases, bothwith and without a hot start. The reactions were incubated at roomtemperature for 30 minutes before amplification.

FIG. 4 is an agarose gel of a pentaplex LATE-PCR without ELIXIR (leftsix lanes), monoplex LATE-PCR with ELIXIR (middle five lanes), andpentaplex with ELIXIR (right six lanes). A molecular weight ladder isshown in far right lane. In the pentaplex reactions, all five targetsare amplified simultaneously.

FIG. 5 shows reaction design (left panels) and dF/dT in the presence ofSybrGreen (right panels) where five primer pairs and (a) one template or(b) one template corresponding to one primer pair+four low copy numberamplicons corresponding to the remaining four primer pairs are included.

FIG. 6 shows pyrosequencing of a LATE-PCR monoplex reaction (left panel)and a multiplex reaction (right panel). The post-LATE-PCR reactions aresplit into five aliquots each and pyrosequencing performed in thepresence of a sequencing primer corresponding to each amplicon.

FIG. 7 shows an embodiment of a Low-T_(m) Probe detection approach.

FIG. 8 compares amplification and detection of a high-T_(m) molecularbeacon probe and a low-T_(m) molecular beacon probe.

FIG. 9 compares resolution of single nucleotide polymorphism inheterozygous CC, heterozygous CT, and homozygous TT using an anneal downprotocol (left) and a melt-up protocol (right).

FIG. 10. LATE-PCR The captions under the bars indicate the temperatureused for RT prior to LATE-PCR. No RTase (light blue bar), theinactivated RTase (blue bar), 30 min at 50° C. (green bar), 30 min at55° C.; 10 min at 60° C.+20 min at 55° C. (orange bar). LATE-PCR isidentical for all samples.

FIG. 11. LATE-PCR protocol with 50 nM limiting primer (LP) and 2 μM RTprimer and LATE-PCR excess primer (XP).

FIG. 12 is a clustal comparison of Influenza Virus M RNA sequences forH3N2, H5N1, H1N1, and B.

FIG. 13 is a clustal comparison of Influenza Virus HA RNA sequences forH3N2, H5N1, H1N1, and B.

FIG. 14 is a clustal comparison of Influenza Virus NA RNA sequences forH3N2, H5N1, H1N1, and B.

FIG. 15 shows a schematic of an embodiment of an assay

FIG. 16 shows the layout and an possible outcomes for an exemplaryassay.

FIG. 17 provides primer, probe, and amplicon sequences that can be usedin an embodiment of an influenza virus assay.

DETAILED DESCRIPTION

All references cited are incorporated herein by reference.

An influenza virus assay can detect and distinguish between varioussub-types and strains of an influenza virus using any suitable nucleicacid amplification technique. This assay can be performed in a singlereaction vessel with all reagents present at the start of an assay. Anassay can use more than one primer pairs in combination with one or moreprobes to amplify and detect specific target nucleic acid sequences ofinfluenza. Using the information obtained from an amplification reactionit is possible to distinguish between various sub-types and strains ofthe an influenza virus. Specifically, an assay can provide a positive ornegative (yes/no) determination of the likely presence or absence ofinfluenza virus types A and B, and sub-types H1N1, H3N2, and H5N1 in asample. An assay also can be used to monitor for one or more mutationsin an influenza virus strain. Mutations in an influenza virus, within,for example the HA and NA, can alter viral infectivity and lethality indifferent hosts and different tissues.

A sample can be any material to be tested, such as, for example, abiological or environmental sample. Biological samples can be obtainedfrom any organism. In one embodiment, a sample is obtained from amammal, such as a human, or a bird. In one embodiment, a samplecomprises a nasopharyngeal aspirate, blood, saliva, or any other bodilyfluid.

A nucleic acid amplification method used in an assay can be a thermalcycling technique, such as a polymerase chain reaction (“PCR”) or anisothermal technique. Standard PCR amplification methods are describedin, for example, U.S. Pat. Nos. 4,683,195 and 4,683,202 (both of whichare incorporated herein by reference). In one embodiment, the nucleicacid amplification method is linear after the exponential (“LATE”) PCR(“LATE-PCR”), as described in U.S. patent application Ser. No.10/320,893, which is incorporated by reference. An embodiment ofLATE-PCR is illustrated in FIG. 1. LATE-PCR is an asymmetric PCR method,which uses unequal concentrations of primers and can yieldsingle-stranded primer-extension products, or amplicons. LATE-PCRamplifications and assays typically include at least 30 cycles, at least60 cycles, or at least 70 cycles. FIG. 2, which shows an agarose gelshowing that amplification product production is less specific when themelting temperature of the Limiting Primer is below that of the ExcessPrimer, demonstrates the specificity advantage that can be afforded by aproperly designed LATE-PCR. An Excess Primer can misprime at a lowannealing temperature, but the reaction can becomes very specific whenthe melting temperature of the Limiting Primer is above the meltingtemperature of the Excess Primer (T_(mL[0])−T_(mX[0])≧0).

As used interchangeably herein, the terms “nucleic acid primer”, “primermolecule”, “primer”, and “oligonucleotide primer” include short, (forexample, between about 16 and about 50 bases) single-strandedoligonucleotides which, upon hybridization with a corresponding templatenucleic acid molecule, serve as a starting point for synthesis of thecomplementary nucleic acid strand by an appropriate polymerase molecule.Primer molecules can be complementary to either the sense or theanti-sense strand of a template nucleic acid molecule. A primer can becomposed of naturally occurring or synthetic oligonucleotides, or amixture of the two. If the primers in a pair of PCR primers are used inunequal concentrations, as is the case in LATE-PCR, primer added at alower concentration is a “Limiting Primer”, and primer added at a higherconcentration is an “Excess Primer.”

As used herein “amplification target sequence” used interchangeably with“target sequence” and “target nucleic acid” refers to a DNA sequencethat provides a template for copying by the steps an amplificationtechnique. An amplification target sequence can be single-stranded ordouble-stranded. If the starting material is RNA, for example messengerRNA, the DNA amplification target sequence is created by reversetranscription of RNA to create complementary DNA, and the amplificationtarget sequence is a cDNA molecule. Thus, in a PCR assay for RNA, ahybridization probe signals copying of a cDNA amplification targetsequence, indirectly signifying the presence of the RNA whose reversetranscription produced the cDNA molecules containing the amplificationtarget sequence. An amplification target sequence typically is bracketedin length by a pair of primers used to amplify it. An extension product(or amplicon), whether double-stranded or, in non-symmetric PCR,single-stranded, is the exponentially amplified amplicon, bracketed bythe primer pair.

An amplification target sequence can be a single nucleic acid sequence.In some cases an amplification target sequence will contain allelicvariations or mutations and, thus, will not be a single sequence, eventhough amplified by a single primer pair. An assay for an amplificationtarget sequence containing variations may use one detection probe forall variations, a single allele-discriminating probe for one variant, ormultiple allele-discriminating probes, one for each variant.

Any suitable primer pair can be used, such as, for example, one of moreof the following primer pairs:

H5 Limiting Primer (reverse complement) GGATAGACCAGCTACCATGATTGCC, Tm= 66.8(H5N1), 21.5(H3N2), 30.3(H1N1), 14.2(B) Excess Primer-RNA 63.2linear GTGGAGTAAAATTGGAATCAATAGG, Tm = 63.6(H5N1), 15.7(H3N2),53.8(H1N1), 34.6(B) H1 Limiting Primer (reverse complement)CACCCGTTTCCTATTTCTTTGGCATTATTC, Tm = 22.5(H5N1), 21.6(H3N2), 66.7(H1N1),30.2(B) Excess Primer, RNA 64.2 linear CCATGACTCCAATGTGAAG, Tm= 36.1(H5N1), 30.1(H3N2), 63.8(H1N1), 20.3(B) N1 Limiting Primer(reverse complement) CAGCACCGTCTGGCCAAGAC, Tm = 69.3(H5N1), 69.3(H1N1),41.0(H3N2), 5.7(B) Excess Primer, RNA 66.3 linear GCAATAACTGATTGGTCAGG,TM = 62.9(H5N1), 62.7(H1N1), 29.9(H3N2), 40.4(B) H3 Limiting Primer(reverse complement) CGTTGTATGACCAGAGATCTATTTTAGTGTCCT, Tm = 12.2(H5N1),67.9(H3N2), 4.6(H1N1), −0.6(B) Excess Primer, RNA 59.7 linearCCATCAGATTGAAAAAGAATTCT, Tm = 29.3(H5N1), 62.7(H3N2), 21.5(H1N1),22.2(B) B(HA) Limiting Primer (reverse complement)CAGGAGGTCTATATTTGGTTCCATTGGC, Tm = 14.4(H5N1), 21.0(H3N2), 24.8(H1N1),67.5(B) Excess Primer, RNA 58.9 linear CGGTGGATTAAACAAAAGCA, Tm = 12.1(H5N1), 26.0(H3N2), 20.3(H1N1), 62.4(B) B(NA) Limiting Primer (reversecomplement) CCCAATACAGGGGACATCACATTTCTTG, Tm = 10.9(H5N1), 16.2(H1N1),−4.6(H3N2), 68.9(B) Excess Primer, RNA 69.7 just linearCATGGGCTGACAGTGAT, Tm = 38.7(H5N1), 43.2(H1N1), 42.5(H3N2), 63.7(B) MLimiting Primer (reverse complement) GGTGACAGGATTGGTCTTGTCTTTAGC, Tm= 67.3(H5N1). 67.3(H3N2), 67.3(H1N1), 14.4(B) Excess PrimerCTAACCGAGGTCGAAAC, Tm = 62.2(H5N1), 62.2(H3N2), 62.2(H1N1), 20.6(B) N2Limiting Primer (reverse complement) GATGCAGCTTTTGCCTTCAACAGAG, Tm= 29.4(H5N1), 16.7(H1N1), 67.4(H3N2), 15.6(B) Excess Primer, RNA 69.4just before hairpin GGTCCAACCCTAATTCCAA, Tm = 13.6(H5N1), 22.0(H1N1),63.4(H3N2), 22.7(B) H3 Control Limiting Primer (reverse complement)CGTTGTATGACCAGAGATCTATTTTAGTGTCCT, Tm = 12.2(H5N1), 67.9(H3N2),4.6(H1N1), −0.6(B) Excess Primer CCATCAGATTGAAAAAGAATTCT, Tm= 29.3(H5N1), 62.7(H3N2), 21.5(H1N1), 22.2(B) H5 Delete Region LimitingPrimer (reverse complement) CCTCCCTCTATAAAACCTGCTATAGCTCCAAA, Tm= 69.7(H5N1), 14.3(H3N2), 20.5(H1N1), 6.5(B) Excess PrimerCGACTGGGCTCAGAAA, Tm = 62.9(H5N1), 17.4(H3N2), 29.3(H1N1), 17.4(B)

In one embodiment all of the above primer pairs are used.

A target sequence can be present at a starting concentration of greaterthan or equal to approximately 1,000,000 copies/sample. In oneembodiment, a target sequence is present at a concentration ofapproximately 10 to approximately 1,000,000 copies/sample. In anotherembodiment a target sequence is present at a concentration of less than10 copies/sample, less than 100 copies/sample, less than 1,000copies/sample, less than 10,000 copies/sample, less than 100,000copies/sample, less than 500,000 copies/sample, or less than 1,000,000copies/sample.

In one embodiment, a target sequence can produce an amplicon as providedin FIG. 17.

An assay includes reagents for an amplification reaction, such as thoseused in LATE-PCR, a symmetric PCR amplification, or an isothermalamplification method. For example, the assay mixture can include each ofthe four deoxyribonucleotide 5′ triphosphates (dNTPs) at equimolarconcentrations, a thermostable polymerase, a divalent cation, and abuffering agent. An assay mixture can include additional ingredients,such as, for example, a separate reverse transcriptase enzyme.Non-natural dNTPs can be used. For instance, dUTP can be substituted fordTTP and used at three-times the concentration of the other dNTPs due tothe less efficient incorporation by Taq DNA polymerase.

An assay also can include reagents that can suppress mispriming. In oneembodiment, a reagent capable of suppressing mispriming is asingle-stranded oligonucleotides capable of forming a stem-and-loopstructure, commonly referred to as a “hairpin” structure such as thosedescribed in, for example, U.S. patent application Ser. No. 11/252,506,(referred to as an “ELIXIR™”), which is incorporated by referenceherein. FIG. 3 demonstrates the ability of an ELIXIR™ to inhibit Taqpolymerase, reducing mispriming and increasing generation of targetproduct. FIG. 4 shows the ability of an ELIXIR™ to reduce mispriming ina multiplex reaction.

An assay also can include an amplicon corresponding to a primer pairthat is capable of suppressing mispriming. In one embodiment, one ormore copies of an amplicon corresponding to a primer pair, where thereare less targets present than primer pairs, is (referred to herein as“mono-multiplex”). For example, an assay can have the capacity ofamplify any one of several different amplicons, but in any particularassay it is possible that either none or only a few viral targetsequences will be present in a sample. Such reactions mono-multiplexreactions can be difficult to construct, because they have to suppressmispriming among the unused primers, while still allowing amplificationof the correct product from any pair of primers. To prevent suchmis-priming, it can be advantageous to add low copy numbers of syntheticamplicons for each primer pair. In one embodiment, approximately 20copies of synthetic amplicons per primer pair can be included in anassay. FIG. 5 demonstrates how inclusion of low levels of syntheticamplicons can suppress mispriming in a mono-multiplex reaction. Thedifference between the design of unsuppressed and “internally suppressedmonomultiplex” reaction is illustrated in FIG. 5 a and the present andabsence of mis-primed products is illustrated in FIG. 5 b.

In another embodiment, reactions that do not contain influenza viralsequences have an internal control that rules out false negatives. Thisinternal control can be observed at a single wavelength. In oneembodiment, the internal control can be observed at 25° C. and can begenerated by amplification of an internal control target possessing avariant of H3 flanked by the H3 primers. Detection can be accomplishedusing any suitable prove, including, for example, a mismatch-tolerantprobe.

Amplification products can be detected by an end-point analysis or usinga real-time analysis. As used herein, the term “real time,” with respectto an amplification reaction, refers to the method by which theamplification reaction is detected. In a “real-time” amplificationreaction, accumulation of amplicon or product is measured during theprogression of the reaction, as opposed to solely after the reaction iscomplete, the latter being “end-point” analysis. In one embodimentdetection is quantitative.

The assays can use any suitable means to detect amplification,including, but not limited to dyes, such as intercalating dyes, DNAbinding agents, and probe molecules. As used interchangeably herein, theterms “nucleic acid probe”, “probe molecule”, and “oligonucleotideprobe” and “hybridization probe” include defined nucleic acid sequencescomplementary to a target nucleic acid sequence to be detected such thatthe probe will hybridize to the target. Probes can be detectablylabeled, such that hybridization of a probe to a target sequence can bereadily assessed. A “detectable label” includes moieties that provide asignal that can be detected and, in some embodiments, quantified. Suchlabels are well known to those in the art and include chemiluminescent,radioactive, metal ion, chemical ligand, fluorescent, or coloredmoieties, or enzymatic groups which, upon incubation with an appropriatesubstrate, provide a chemiluminescent, fluorescent, radioactive,electrical, or calorimetric signal. Methods of detection of such signalsare also well known in the art.

Probes can be composed of naturally occurring or syntheticoligonucleotides and include labeled primers. Some hybridization probes,for example molecular beacon probes, emit an increased detectable signalupon hybridizing to their complementary sequence without enzymaticaction to hydrolyze the probes to generate a signal. We refer to suchprobes as probes that hybridize to their target and “signal uponhybridization.” Other probes, for example TaqMan™. dual fluorescentlylabeled random coil probes are cut, or hydrolyzed, during theamplification reaction, and hydrolysis leads to a detectable signalchange. Probes that rely on hydrolysis as part of signal generation arenot probes that “signal upon hybridization.”

In one embodiment, an assay uses a “molecular beacon probe,” which is asingle-stranded oligonucleotide, typically 25-35 bases-long, in whichthe bases on the 3′ and 5′ ends are complementary. Molecular beaconprobes are discussed in, for example, U.S. Pat. Nos. 5,925,517,6,037,130, 6,103,476, 6,150,097, and 6,461,817, and U.S. Patent Appl.Pub. No. 2004/0023269A1, all of which are incorporated by reference. Amolecular beacon probe can form a hairpin structure at temperatures atand below those used to anneal the primers to the template (typicallybelow about 60° C.). The double-helical stem of the hairpin brings afluorophore attached to one end (often, but not necessarily the '5 end)of a probe in proximity to a quencher attached to the other end of theprobe (typically, but not necessarily, the 3′ end). In the hairpinconfiguration, probe fluorescence is quenched. If a probe is heatedabove the temperature needed to melt the double stranded stem apart, ora probe is allowed to hybridize to a target oligonucleotide that iscomplementary to a sequence within the single-strand loop of a probe,fluorophore and quencher are separated, and the resulting conformationshows increased fluorescence. The strength of a fluorescent signal canincreases in proportion to the amount of a molecular beacon hybridizedto an amplicon. Molecular beacons with different loop sequences can beconjugated to different fluorophores in order to monitor increases inamplicons that differ by as little as one base (Tyagi, S. and Kramer, F.R. (1996) “Molecular Beacons: Probes That Fluoresce Upon Hybridization,”Nat. Biotech. 14:303-308; Tyagi, S. et al., (1998) “Multicolor MolecularBeacons for Allele Discrimination.” Nat. Biotech. 16: 49-53; Kostrikis,L. G. et al., (1998) “Spectral Genotyping of Human Alleles,” Science279: 1228-1229).

Any suitable fluorophore/quencher pair can be used in a molecular beaconprobe. In one embodiment, four probes are used each with a singlefluorophore, wherein the flourophores are texas red, CY3, CY5, and FAM.Any suitable quencher can be used, such as, for example, Black Hole™quenchers, dabsyl, and BHQ1. In one embodiment, an assay include one ormore fluorophore/quencher pair, wherein the pair can be any of texasred/dabsyl, CY5/dabsyl, FAM/dabsyl, CY5/BHQ1, and CT3/dabsyl. In anotherembodiment, an assay uses one or more of the following probes:

H5 Texas Red-CGCGACTAGGGAACTCGCTCGCG -Dabsyl, Tm = 52.7(H5N1),8.0(H3N2), 24.9(H1N1), 13.0(B) H1CY3-CGCGGATTGGCTTTTTACTTTCTCACCGCG-Dabsyl, Tm = 27.8(H5N1), 20.1(H3N2),56.6(H1N1), 12.7(B) N1 FAM- GGCGGATGCTGCTCCCACTACCGCC -Dabsyl, Tm= 56.3(H5N1), 56.3(H1N1), 12.1(H3N2), 25.8(B) H3CY5-CGCTGAAAGCGTTTCTCGAGGTCCTG-BHQ1, Tm = 9.9(H5N1), 54.5(H3N2),15.4(H1N1), 9.1(B) B(HA) Beacon Probe 1 Texas Red-GCGAGTTTGCATGTTCTCCTGTCTCGC -Dabsyl, Tm = 19.2(H5N1), 16.2(H3N2),15.3(H1N1), 52.1(B) Beacon Probe 2 CY5- GCGAGTTTGCATGTTCTCCTGTCTCGC-Dabsyl, Tm = 19.2(H5N1), 16.2(H3N2), 15.3(H1N1), 52.1(B) B(NA) BeaconProbe 1 Texas Red- GCCGCTCCATTGAAACCATTACGCGGC -Dabsyl, Tm = 26.3(H5N1),27.9(H1N1), 21.2(H3N2), 53.1(B) Beacon Probe 2 ( CY-GCCGCTCCATTGAAACCATTACGCGGC -Dabsyl, Tm = 26.3(H5N1), 27.9(H1N1),21.2(H3N2), 53.1(B) M CY3- GCGCTATAGAGAGAACAGCGC -Dabsyl, Tm = 33.8(H5N1), 33.8(H3N2), 33.8(H1N1), 9.6(B) N2 FAM- GGCCGCCTATTACCTCTCGGCC-Dabsyl, Tm = 30.0(H5N1), 27.7(H1N1), 38.9(H3N2), 20.6(B) H3 ControlCY5- CGCTGAAAGCGTTTCTCGAGGTCCTG -BHQ1, Tm = 32.9 vs. modified ampliconsequence CAGGAACTCTAGAAA H5 Delete RegionFluor-stemTCCTCTCTTTTTTCTTCTTCTCTstem-Dabsyl, Tm = 58.9(H5N1),−3.8(H3N2), 20.4(H1N1), 13.4(B)

In another embodiment, all of the above molecular beacon probes are usedin an assay. In a further embodiment, an assay can include ninesequence-specific molecular beacons that are capable of detecting seveninfluenza virus targets. In a further embodiment, three molecularbeacons probes, each detectable at a different wavelength, can form aprobe-target hybrid at T_(m) 45° C. and two additional molecular beaconprobes, each detectable at a different single wavelength, can form aprobe-target hybrid at T_(m) 30° C. Further, two additional molecularbeacon probes, each detectable a two different wavelengths, can form aprobe-target hybrid at T_(m) 45° C. An additional mis-match tolerantprobe can also be used to detect one of the viral targets at 40° C. anda variant of that sequence present in an internal control at 25° C.

In one embodiment, an assays can use a “Low-T_(m) Probe.” A Low-T_(m)Probe is discussed in U.S. patent application Ser. No. 10/320,893 andrefers to a labeled hybridization probe that signals upon hybridizationto its target, which in a LATE-PCR is the Excess Primer-Strand generatedby extension of the Excess Primer, and that has a T_(m[0]) ^(P) at least5° C. below or at least 10° C. below the T_(m[0]) of the primer thathybridizes to and extends along the Excess Primer-Strand, which in aLATE-PCR is the Limiting Primer. FIG. 7 shows an embodiment of aLow-T_(m) Probe detection approach.

As shown in FIG. 8, Low-T_(m) Probes can be more specific over a widertemperature range and can display lower backgrounds. Low-T_(m) Probesalso show less amplification at higher concentrations than High-T_(m)Probes.

In another embodiment, an assay can use a “Super-Low-T_(m) Probe.” Thisprobe also is discussed in U.S. patent application Ser. No. 10/320,893.

An assay can include more than one probe. In one embodiment, multiplemolecular beacon probes are used. In another embodiment, the probes arecapable of forming probe-target hybrids are more than one temperature.In a further embodiment, multiple probes can be used, where a firstprobe forms a probe-target hybrid at a first temperature and a secondprobe forms a probe-target hybrid at a second temperature. In oneembodiment, five molecular beacon probes can form a probe-target hybridat a temperature of greater than 45° C. and can be detected at 40° C.and two molecular beacon probes can form a probe-target hybrid at 30° C.and can be detected at 25° C.

An assay can also include mismatch tolerant probes, such as, forexample, fluorescent probes. In one embodiment, an assay uses a mismatchtolerant probes. An assay also can detect probe-target hybrids as asample is cooled after PCR amplification (“anneal down”). This approachcan be used in end-point fluorescence detection. This anneal-downapproach can be more sensitive and provide better resolution thancooling first and then reading during warm-up (melt-up), because theread-during-cooling approach can minimize formation of hairpinstructures in a target sequence. FIG. 9 compares resolution of singlenucleotide polymorphism in heterozygous CC, heterozygous CT, andhomozygous TT using an anneal down protocol (left) and a melt-upprotocol (right). In one embodiment, the temperature of an assayreaction is changed from less than 95° C. to less than 65° C. than 45°C., to less than 25° C.

An influenza viral assay (or an assay of any RNA virus) can involvereverse transcription (RT) as a first step of a detection reaction.During reverse transcription RNA sequences are converted tocomplementary DNA (cDNA), providing a cDNA template for PCRamplification.

An approach to RT-PCR is the use of a “One-Step RT-PCR system.” In asystem of this type, reagents for both RT and PCR can be added to asample in a single mixture and the reaction tube can be sealed andplaced in a thermocycler. The RT and PCR enzyme-catalyzed reactions arecarried out sequentially in the thermocycler, taking advantage of thedifferent thermostabilities of the enzymes involved (typically, areverse transcriptase and a thermostable DNA polymerase) and by settingan appropriate thermal profile. An initial incubation at non-denaturingtemperature allows RT to occur first. The temperature then can be raisedto initiate PCR; at this temperature, the reverse transcriptase can beinactivated, but the DNA polymerase is not. When a “hot start” is used,DNA polymerase is kept inactive during the RT step by interaction with aspecific antibody. When the temperature is elevated, the antibody can bedenatured and the DNA polymerase activated. In one embodiment, amulti-functional enzyme, having both a RTase activity and a DNApolymerase activity, can be used. In a further embodiment, amulti-functional enzyme having RTase activity, DNA polymerase activity,and exonuclease activity can be used, where the exonuclease activity cancleave double-stranded DNA in TaqMan-type detection method.

The temperature and duration of RT and PCR steps can be readilydetermined by one of skill in the art. In one embodiment, an RT step canbe performed for from less than 2 minutes to more than 60 minutes at atemperature of from approximately 50-60° C. and a DNA-polymerase stepcan be performed as a thermocycle at approximately 95° C. for severalcycles as discussed elsewhere.

An assay can be performed using any suitable device, such as a thermalcycler. In one embodiment, an assay is performed using a portabledevice, a man-portable device, or a handheld device, such as, forexample, a Bioseeq II. In another embodiment, an assay is performedusing a bench-top device, including, for example, an ABI Prism 7700Sequence Detector (Applied Biosystems, Inc., CA) machine, a CepheidSmart Cycler, and a Primus PCR thermocycle.

An assay can be performed in less than or equal to 30 minutes, less thanor equal to 20 minutes, less than or equal to 15 minutes, or less thanor equal to 10 minutes.

Assay reagents can be provided as a kit or a consumable. The reagentscan be supplied as a lyophilized preparation. Each reagent can besupplied separately or as a mixture of one or more reagents. Reagentsalso can be supplied on a substrate, such as a bead. A lyophilizedreagent can be stable for more than one year.

An assay can yield single-stranded products for further use, for exampleas starting material for DNA sequencing or as probes in other reactions,or can be used in other assays. In one embodiment, single stranded DNAproduced by an assay (assay product) can be sequenced using any suitablesequencing method, such as, for example, the dideoxy-method orpyrosequencing (Salk et al. (2006) Anal. Biochem. 353:124, incorporatedby reference) by diluting a fraction of the assay reaction products intoa sequencing reaction mixture. Assay product can be diluted byapproximately 1:10, approximately 1:20, approximately 1:50,approximately 1:100, or approximately 1:200 or more for use in asequencing reaction. FIG. 6 shows a LATE-PCR multiplex reaction, inwhich one sample is split into five aliquots each spiked with adifferent sequencing primer, and sequenced.

An assay can distinguish Influenza Type B and Type A virus. In oneembodiment, an assay distinguishes Influenza Type B and Type A virus onthe basis of sequences in the HA and NA genes. Within the Type A virusesan assay can distinguish between subtypes H5 (with or without N1 or N2),H1 (with or without N1 or N2), and H3 (with or without N1 or N2). In oneembodiment, an N1 target sequence used is conserved for the H5 and H1subtypes and can be useful for detecting H5N1 and H1N1. In anotherembodiment, H3N1 can be determined and such a determination can indicateviral reassortment. The N2 target sequence used is characteristic of theH3N2 subtype, thus, detection of H5N2 or H1N2 can indicate viralreassortment. FIGS. 12-14 shows clustal comparisons of influenza virusM, HA, and NA proteins for virus H1N1, H5N1, H3N2, and B. Such analysisis useful in interpreting data obtained from an assay and fromsubsequent sequencing of assay products. Using information obtainingfrom an assay, it is possible to monitor mutations in a known virusstrain, which allows for detection of and prediction of changes invirulence and infectivity.

An exemplary avian influenza assay and possible results of thisexemplary assay are provided in Table I and FIG. 15. FIG. 16 shows aschematic of an embodiment of an assay and FIG. 17 provides primer,probe sequences, and amplicon sequences that can be used in anembodiment of an influenza virus assay. In one embodiment, an assayinclude all of the primers and probes of FIG. 17 in a mono-multiplexassay. The features of such a mono-multiplex are summarized in Table Iand the 15 possible outcomes of the reaction are illustrated 16.

TABLE I Position Amplicon Primers Target Sequence Probe Type Color(s)Melting Tm 1 H5 H5 M. Beacon Red 45 C. 2 H1 H1 M. Beacon Yellow 45 C. 3N1 N1 M. Beacon Green 45 C. 4 H3 H3 EXO-R Blue 45 C. 6 Type A M gene Mgene M. Beacon Yellow 30 C. 7 N2 N2 M. Beacon Green 30 C. 4/5 Type B HAonly Type B HA only M. Beacon Blue 45 C. Type B HA only Type B HA onlyM. Beacon Red 45 C. 4/5 Type B NA only Type B NA only M Beacon Blue 45C. Type B NA only Type B NA only M. Beacon Red 45 C. 4/5 Type B HA + NAType B HA + NA M Beacon Blue + Blue 45 C. Type B HA + NA Type B HA + NAM. Beacon Red + Red 45 C. 8 H3 int. control mis-matched H3 EX0-R Blue 30C. H5 int. control no matches no probe H1 int. control no matches noprobe N1 int. control no matches no probe N2 int. control no matches noprobe M int. control no matches no probe Type B HA i.c. no matches noprobe Type B NA i.c. no matches no probe The exemplary assay describedin this table contains: 8 pairs of primers 3 Molecular Beacons with 45°C. 2 Molecular Beacons with 30° C. 2 pairs of Molecular Beacons both at45° C. Total = 9 Molecular Beacons 1 mismatch-tolerant prove 1detectable internal control 7 undetected internal controls

EXAMPLE 1

Starting with samples of purified RNA, the HA RNA (1770 nucleotideslong) and the NA RNA (1400 nucleotides long) are both be reversetranscribed in toto using random hexamers in a highly efficient two stepRT-procedure. Each reaction also contains low levels of an M-Genecontrol DNA. The resulting control and cDNA molecules are amplified intwo parallel multiplex LATE-PCR assays that each generate six amplicons.The presence of Eurasian H5N1 strain in a sample is established byprobing for M, N1 and two different H5 sequences that are likely to becrucial for human-to-human transmission and for virulence. Reactionsthat do not generate either signal for H5 Eurasian will neverthelessproduce a control DNA signal, proving that they are not false negatives.Reactions that do signal the presence of the H5 Eurasian strain fromeither of two independent probes (one in Multiplex A and one inMultiplex B) also generates a strong M-gene signal in both Multiplex Aand Multiplex B. However, some samples may generate a signal for an Mprotein and only one of two possible HA signals. This is regarded as anindication of viral evolution. All samples that generate either one ortwo HA signals, or an N1 signal plus an M-gene signal are immediately beprocessed further for analysis. The amplicons for the all portions of HAand NA already are present in the LATE-PCR multiplex reactions. All 10HA and NA amplicons are 300-500 bp in length and are processed forparallel pyrosequencing sequencing.

EXAMPLE 2

This example is directed to an RT-LATE-PCR assay for the quantificationof Oct4-specific sequences in embryonic mouse cells. Oct4 is a geneexpressed in totipotent and pluripotent cells and, therefore,preimplantation embryos contain considerable levels of Oct4 RNA. Inaddition, each cell contains two copies of the Oct4 gene (Oct4 genomicDNA). In the experiments presented in this example, Oct4 RNA and DNA areco-purified and co-quantified, according to a method previouslypublished by this laboratory (Hartshorn C, Anshelevich A, Wangh L J.Rapid, single-tube method for quantitative preparation and analysis ofRNA and DNA in samples as small as one cell. BMC Biotechnol 2005; 5:2.).Briefly, single embryos at the 8-cell stage are transferred to tinydroplets of dry lysing reagents placed on the lid of PCR tubes. Aftercell lysis, which occurs very rapidly, the tube is placed on the lid andinverted. One-step RT-PCR is performed by adding the reagents in thesame tube already containing the lysed sample. Thus, both RNA and DNAare present in each sample. LATE-PCR primers are designed within anexon, also according to our published strategy, so that ampliconsgenerated by Oct4 cDNA molecules and Oct4 genomic DNA molecules areidentical and detected by the same fluorescent probe, asequence-specific molecular beacon conjugated to the TET fluorescentdye. The final volume of these assays is 50 μl, according to theinstruction of the One-Step RT-PCR kit, but the volume can be decreasedto 25 μl.

The RT-LATE-PCR reaction is carried out in an ABI Prism 7700 SequenceDetector (Applied Biosystems, CA) and quantification of “total DNA”(cDNA+genomic DNA) copy numbers for each sample is achieved bycomparison with standard scales prepared with serial dilutions ofcommercially available genomic DNA. (Because the Oct4 primers aredesigned to amplify equally cDNA and genomic DNA, standard scales ofgenomic DNA are amplified exactly with the same efficiency as unknowncDNA samples, ensuring accurate quantification.) The total number ofOct4 templates in each 8-cell embryo includes 16 copies of genomic DNA(two copies of the gene per cell, one on each chromosome 17) while allthe other copies are due to the presence of cDNA and, thus, reflect theOct4 RNA content of the embryo.

Several One-Step RT-PCR kits are tested using this assay and theefficiencies for Oct4 RNA quantification are compared. FIG. 10 shows theeffect of temperature on SuperScript III reverse transcriptase(Invitrogen).

The blue and orange bars in the figure are comparable to the light blue“No RT” bar, indicating that under these temperature conditions RT doesnot take place and only genomic Oct4 DNA is amplified in the samples (16copies per embryo, as expected). At 55° C. (green bar), however, RToccurs and cDNA is efficiently generated. Considering that the reversetranscriptase used for these experiments is active in the 42-60° C.range, this narrow window of activity is unexpected. To clarify thispoint, the thermodynamics of the Oct4 primers during RT is analyzed andcompared to their behavior during PCR.

Visual OMP 5.0 software(VOMP) is used for this analysis and the resultsare summarized in FIG. 11. The two primers used for a LATE-PCR assay(limiting primer, LP, and excess primer, XP) have different T_(m)s andconcentrations. In the Oct4 RT-LATE-PCR assay the most abundant primer(XP) is also the RT primer, being antisense to Oct4 RNA. As shown inFIG. 11, the effective T_(m) of this primer calculated in the presenceof double-stranded DNA during the PCR annealing step (at 55° C.) is of66° C., very close to the calculated T_(m) of 67° C. The effective T_(m)of this same primer, however, drops dramatically to 53° C. in thepresence of single-stranded RNA, even if the temperature of RT is alsoset at 55° C. This change results in a much lower percent of primerhybridized during RT than during the PCR annealing step, although thetemperature is the same during the two steps. Although only 50% of theavailable primer is hybridized to target at 55° C., the primer waspresent at high concentration (2 μM) which allowed efficient RT.Additional VOMP analyses show that Oct4 XP's effective T_(m) during RTdoes not change in the 50-60° C. range, which explains why increasing RTtemperature in this case led to RT failure (much less primer was boundto target at 60° C. than at 55° C. and, contrary to the manufacturer'sindications, the RTase was completely denatured after the initial 10minutes at 60° C., see next section). On the other hand, the failure ofRT at temperature lower than 55° C. (when, based solely on T_(m), moreprimer should be hybridized) is probably due to increased levels ofsecondary structure of the target RNA interfering with the ability ofthe reverse transcriptase to progress along the template strand.

These results indicate that primers designed for PCR or LATE-PCR alsoshould be analyzed in terms of their thermodynamic modification of aprimer's design so that its T_(m) can meet the necessary requirementsduring both RT and PCR. In cases where this is not possible due torestraints intrinsic to the sequence, a third primer—designed tohybridize only during the RT step—could be added to the one-stepmixture. In addition, the characteristics of LATE-PCR are advantageousto promote RT priming in a one-step assay. In fact, by designating theXP to be also the RT primer we are able to use higher RT primerconcentrations than those used under standard conditions in RT-PCRassays.

EXAMPLE 3

This example demonstrates optimization of RT reaction parameters.

Satisfactory RT results are obtained for two different genes shorteningthe RT step from 30 to 5 minutes, although a slight loss of sensitivityis observed. Further reducing RT to 2 or 3 minutes still yieldsacceptable results. The reverse transcriptase used was SensiScript byQiagen. (Raja et al., 2002. Temperature-controlled Primer Limit forMultiplexing of Rapid, Quantitative Reverse Transcription-PCR Assays:Application to Intraoperative Cancer Diagnostics. Clinical Chemistry48:8, 1329-1337.)

RT also performed with SuperScript II (Invitrogen) for 5 minutes. (Rajaet al., 2005. Technology for Automated, Rapid, and Quantitative PCR orReverse Transcription-PCR Clinical Testing. Clinical Chemistry 51:5,882-890.

RT is successfully carried out for just 1 minute with either MMLV(Moloney Murine Leukemia Virus RT) or SuperScript III (Stanley andSzewczuk, 2005. Multiplexed tandem PCR: gene profiling from smallamounts of RNA using SYBR Green detection. Nucleic Acids Research 33:20,e180.)

Based on the above studies, a One-Step RT-PCR assay for detection ofavian flu is designed that will encompass a RT step of no more than 5minutes and as low as 1 minute. In doing so, we are aware that theoptimal length of the RT reaction depends on several factors, including,but not limited to, efficient primer binding (see Example 2). Theintrinsic thermostability of the RT enzyme also comes into play whenchoosing the temperature for RT, because the half-life of any enzymesharply decreases at increasing temperatures, although some enzymes aremore stable than others.

A clear example is provided by the following table posted on the web bythe manufacturer Invitrogen

Summary of RT Half lives at 50, 55, and 60° C.

Temperature Superscript ™ II RT (min) Superscript ™ III RT (min) 50° C.6.1 220 55° C. 2.2 24 60° C. ND 2.5

From this table it follows that, when working with SuperScript III at60° C. or with SuperScript II at 55° C., the optimal RT step duration isno more than 5 minutes in any case, independently from the primerT_(m)s, because the enzyme is completely denatured in this period oftime.

Newer RTases with broader thermostability ranges are commerciallyavailable. For example, StrataScript 5.0 from Stratatgene, has ahalf-life of 35 minutes at 55° C. There is also a number of polymerasescommercialized by Roche and derived from thermophilic bacteria, whichhave both RTase and DNA polymerase activity.

We note that it is important to designing gene-specific RT primers withT_(m)s precisely calculated for optimal hybridization to target at atemperature elevated enough to minimize the secondary structure ofsingle-stranded, GC-rich RNA molecules such as those present in viralgenomes, but at the same time allowing a sufficient half-life of thechosen enzyme. The importance and the subtleties of this approach arenot widely recognized, as shown by the suggestion: “Primers forreal-time RT-PCR should be designed according to standard PCRguidelines” (Platinum Quantitative RT-PCR ThermoScript One-Step Systeminstruction sheet, included with product purchased in 2006).

EXAMPLE 4

This example demonstrates the use of a Smiths Detection Bio-Seeq IIinstrument as a portable, point-of-care assay device. The Bio-Seeq IIused in this example is comprised of four independently operatingthermal cycling units, each encasing a long thin-walled sample tubehaving a sample volume of 25 ul. The primers and probes provided in FIG.17 are used. Each sample is viewed using four-color fluorescence opticsfor dyes that emit at 520 nm, 580 nm, 625 nm, 680 nm. All colors can beviewed simultaneously without moving parts, a feature of the BioSeeqthat reduces sampling time and lowers the risk of mechanical failure. Tomake full use of the broad detection temperature range available inLATE-PCR each unit can ramp up at 10° C./sec between 25-95° C., and isactively cooled at a rate of at least 2.5° C./sec between 95-25° C. Thetolerance for thermal variance at any chosen hold temperature is ±1° C.The unit is AC or battery powered.

Each of the LATE-PCR mono-multiplex assays described below is designedto detect and distinguish any one of 15 possible outcomes in a singleclosed-tube. See FIGS. 15 and 16. These assays are easy to use “in thefield” and provide rapid definitive yes/no answers as to the absence orpresence of Influenza Virus sub-types H1N1, H3N2, B and H5N1. The assaysalso detect the presence of a Type B virus, or Type A influenza virus ofunknown sub-type and include internal controls to rule out falsenegatives.

Each mono-multiplex reaction includes internal control target sequences,as shown in FIG. 17, at low copy number (approximately 20) to insurethat all primer pairs are engaged in amplifying either a viral targetsequence or an internal control. Accordingly, the reaction describedbelow utilizes eight pairs of primers and eight internal controls.

Each mono-multiplex reaction is read at end-point by dropping thetemperature to 40° C. and then to 25° C. Nine sequence-specificmolecular beacons are used in this reaction to detect 7 of the possibleviral targets. Three molecular beacons, each of a single color, formprobe-target hybrids at T_(m) 45° C. Two additional molecular beacons,each of a single color, form probe-target hybrids at T_(m) 30° C. Twoadditional molecular beacons, each with two colors, form probe-targethybrids at T_(m) 45° C.

One mis-match tolerant probe is used to detect one of the viral targetsat 40° C. and a variant of that sequence present in an internal controlat 25° C. Seven of eight internal controls go undetected because theypossess no targets for any probe.

Each mono-multiplex reaction is designed to distinguish Influenza Type Band Type A viruses on the basis of sequences in the HA and NA genes.Within the Type A viruses the reaction distinguishes between subtypes H5(with or without N1 or N2), H1 (with or without N1 or N2), and H3 (withor without N1 or N2). The N1 target sequence used is conserved for theH5 and H1 subtypes and therefore is useful for detecting H5N1 and H1N1.Detection of H3N1 would indicate viral reassortment. The N2 targetsequence used is characteristic of the H3N2 subtype. Therefore detectionof H5N2 or H1N2 would indicate viral reassortment. The mono-multiplexreaction described here is a one-step RT-LATE-PCR reaction. The chemicalfeatures of this one-step process are described elsewhere.

One assay provides a reliable means of detecting the Eurasian subtype ofH5. Specimens that test positive for H5 Eurasian in the field are besent to an analytical laboratory for complete multiplex analysis andsequencing.

In a second assay, the H5 amplicon produced in the field includes theregion of the RNA known to code for high pathogenicity of the Eurasiansub-type. This region is less conserved, but very important. Under thesecircumstances the tube that tests positive in the field for Eurasian H5is sent to the laboratory for immediate sequencing of the H5 amplicon.There is no need to transport the specimen itself or additionalamplification The next step involves in-depth laboratory analysis ofinfluenza genes using LATE-PCR multiplexing and nucleic acid sequencing.Starting with samples of purified RNA, the HA RNA (1770 nucleotideslong) and the NA RNA (1400 nucleotides long) are both be reversetranscribed in toto using random hexamers in a highly efficient two stepRT-procedure. Each reaction also contains low levels of the same M-Genecontrol DNA describes for the BioSeeq POC assays above. The resultingcontrol and cDNA molecules are amplified in two parallel multiplexLATE-PCR assays that each generate six amplicons (FIG. 15).

The possible presence of Eurasian H5N1 strain in a sample will beestablished by probing for M, N1 and two different H5 sequences whichare likely to be crucial for human-to-human transmission and forvirulence. Reactions that do not generate either signal for H5 Eurasianstill produce a Control DNA signal, proving that they are not falsenegatives. Reactions that do signal the presence of the H5 Eurasianstrain from either of two independent probes (one in Multiplex A and onein Multiplex B) also generate a strong M-gene signal in both Multiplex Aand Multiplex B. However, some samples may generate a signal for theMatrix protein and only one of the two possible HA signals. This isregarded as an indication of viral evolution. All samples that generateeither one or two HA signals, or an N1 signal plus an M-gene signal willimmediately be processed further for analysis. Amplicons for the allportions of HA and NA are already be present in the LATE-PCR multiplexreactions. All 10 HA and NA amplicons are 300-500 bp in length and areprocessed for parallel pyrosequenceing as described above.

1. An assay comprising more than one primer pair and more than onedetection probe, a low copy number synthetic amplicon corresponding toeach of the primer pairs, wherein the primer pairs are selected from thegroup consisting of: a) GGATAGACCAGCTACCATGATTGCC, (SEQ ID NO: 1) andGTGGAGTAAAATTGGAATCAATAGG; (SEQ ID NO: 2) b)CACCGGTTTCCTATTTCTTTGGCATTATTC, (SEQ ID NO: 3) and CCATGACTCCAATGTGAAG;(SEQ ID NO: 4) c) CAGCACCGTCTGGCCAAGAC, (SEQ ID NO: 5) andGCAATAACTGATTGGTCAGG; (SEQ ID NO: 6) d)CGTTGTATGACCAGAGATCTATTTTAGTGTCCT, (SEQ ID NO: 7) andCCATCAGATTGAAAAAGAATTCT; (SEQ ID NO: 8) e) CAGGAGGTCTATATTTGGTTCCATTGGC,(SEQ ID NO: 9) and CGGTGGATTAAACAAAAGCA; (SEQ ID NO: 10) f)CCCAATACAGGGGACATCACATTTCTTG, (SEQ ID NO: 11) and CATGGGCTGACAGTGAT;(SEQ ID NO: 12) g) GGTGACAGGATTGGTCTTGTCTTTAGC, (SEQ ID NO: 13) andCTAACCGAGGTCGAAAC (SEQ ID NO: 14) h) GATGCAGCTTTTGCCTTCAACAGAG, (SEQ IDNO: 15) and GGTCCAACCCTAATTCCAA; (SEQ ID NO: 16) f)CGTTGTATGACCAGAGATCTATTTTAGTGTCCT, (SEQ ID NO: 7) andCCATCAGATTGAAAAAGAATTCT; (SEQ ID NO: 8) and g)CCTCCCTCTATAAAACCTGCTATAGCTCCAAA, (SEQ ID NO: 17) and CGACTGGGCTCAGAAA;(SEQ ID NO: 18) and

wherein the detection probe is a molecular beacon prove selected fromthe group consisting of: a) Texas Red-CGCGACTAGGGAACTCGCTCGCG (SEQ IDNO: 19)-Dabsyl, b) CY3-CGCGGATTGGCTTTTTACTTTCTCACCGCG (SEQ ID NO:20)-Dabsyl, c) FAM- GGCGGATGCTGCTCCCACTACCGCC (SEQ ID NO: 21)-Dabsyl, d)CY5-CGCTGAAAGCGTTTCTCGAGGTCCTG (SEQ ID NO: 22)-BHQ1, e) Texas Red-GCGAGTTTGCATGTTCTCCTGTCTCGC (SEQ ID NO: 23)-Dabsyl, f) CY5-GCGAGTTTGCATGTTCTCCTGTCTCGC (SEQ ID NO: 23)-Dabsyl, g) Texas Red-GCCGCTCCATTGAAACCATTACGCGGC (SEQ ID NO: 24)-Dabsyl, h) CY5-GCCGCTCCATTGAAACCATTACGCGGC (SEQ ID NO: 24)-Dabsyl, i) CY3-GCGCTATAGAGAGAACAGCGC (SEQ ID NO: 25)-Dabsyl, Tm = 33.8 j) FAM-GGCCGCCTATTACCTCTCGGCC (SEQ ID NO: 26)-Dabsyl, k) CY5-CGCTGAAAGCGTTTCTCGAGGTCCTG (SEQ ID NO: 22) -BHQ1, and l)Fluor-stemTCCTCTCTTTTTTCTTCTTCTCT (SEQ ID NO: 28)stem-Dabsyl.